All ETDs from UAB

Advisory Committee Chair

Moon H Nahm

Advisory Committee Members

William H Benjamin, Jr

David E Briles

Suzanne M Michalek

Hui Wu

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that expresses a capsular polysaccharide (PS) to shield underlying antigenic structures from complement-mediated opsonophagocytosis. Thus, capsular PS is the major virulence factor, the target of primary immune responses to pneumococcal infections, and the antigenic component of pneumococcal vaccines. Pneumococci have diverse capsular PS synthesis (cps) loci and express over 90 different capsule types. Such diversity helps pneumococci escape immune detection and adapt to changing environments. Recombination of cps genes has been recognized as the main driver of capsule diversity. However, we show point mutations in cps genes encoding glycosyltransferases can broaden enzymes' substrate specificities and permit biosynthesis of hybrid capsules comprised of two different repeating units (RU). One example was discovered in serogroup 6, which contains serotypes 6A-D, that are serologically, biochemically, and genetically distinct. In this thesis, we characterized two novel hybrid serogroup 6 variants, named serotypes 6F and 6G, whose capsule structures were a mixture of 6A and 6C, or 6B and 6D, PSs, respectively. Serotype 6F had the rhamnosyltransferase gene wciP&alpha (like 6A and 6C), 6G had wciP&beta (like 6B and 6D), but both had the &alpha1,3-galactosyltransferase gene wciN&alpha (like 6A and 6B) containing a single mutation (Ala150Thr) in a conserved NXG motif associated with enzyme substrate specificity. Site-directed mutagenesis studies showed that WciN&alpha (Ala150Thr) was responsible for the hybrid serotype. Another example was found in serogroup 11, containing serotypes 11A-F, whose RUs have four hexoses. The fourth hexose is glucose in 11A and 11E, and N-acetylglucosamine in 11B, 11C and 11F. Our study found serotype 11D PS was a hybrid containing both glucose and N-acetylglucosamine as the fourth hexose in 75% and 25% of its RUs, respectively. Genetic studies found that residue 112 in WcrL was asparagine in 11A and 11E, alanine in 11B, 11C and 11F, and serine in 11D. Site-directed mutagenesis confirmed that residue 112 mediated substrate specificity, possibly by steric hindrance in the binding pocket, and that the 11D WcrL was bi-specific for both donor substrates. Thus, minimal modifications of an enzyme's active site can alter pneumococcal capsule structure by expanding its catalytic specificity.

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